The invention relates to the detection of nucleic acids associated with disease states. In particular, the invention provides for the detection of nucleic acids in acellular biological fluids as diagnostic assays for chronic illnesses and infectious diseases. Also provided are therapeutic approaches to treating chronic illnesses.
Chronic diseases such as cancer, autoimmune diseases, chronic fatigue syndrome and the like afflict millions of people throughout the world. It is known that environmental and other factors (e.g., genotoxic compounds, infectious retroviruses, retroelements and the like) can directly disrupt and/or damage DNA and may play a role in the development of a number of chronic illnesses. The mechanisms by which damage to genetic material leads to the onset of these diseases is not well understood, however. It is known that certain sites in the genome (e.g., fragile sites) are particularly susceptible to such modifications. For instance, it is known that the distribution of insertion sites for retroviruses and retroelements is not random and that fragile sites are often preferred (see, e.g., Craigie Trends in Genetics 8:187 (June 1992); De Ambrosis et al. Cancer Genet. Cytogenet. 60:1-7 (1992); Durnam et al. and Romani et al. Gene 135:153-160 (1993)).
Fragile sites themselves are associated diseases. For instance, expansion of long of blocks of repeated CCG triplets together with methylation of CpG islands in particular fragile sites on the X chromosome have been linked to the fragile X syndrome, an inherited mental retardation (see, e.g., Sutherland and Richards, Proc. Nat. Acad. Sci. USA 92:3636-3641 (1995).
The detection of nucleic acids from pathogens such as bacteria, parasites and viruses, is a commonly used method for diagnosis of disease. For instance, detection of viral sequences is useful in diagnosis of disease. Enteroviruses are a heterogeneous group of human pathogens and opportunistic agents responsible for a broad spectrum of diseases and make up a large genus within the family Picornaviridae. The genus includes polioviruses, coxsackieviruses, echoviruses as well as a number of uncharacterized enteroviruses isolated from humans and other primates. For a review of taxonomy of Picornaviridae see, Virus Taxonomy: Classification and Nomenclature of Viruses Murphy et al., eds (Springer Verlag, 1995).
Like other members of the picornaviridae, enteroviruses are small, single-stranded, nonenveloped RNA viruses. Enteroviruses are distinguished from other members of the picornaviridae by their stability in acid and their fecal-oral route of passage and transmission.
Polioviruses (which exist as at least three serotypes) are the most clinically significant of the enteroviruses worldwide, causing paralytic disease in children in developing countries. Non-polioenteroviruses (NPEV) are also responsible for large numbers of symptomatic infections each year. They are the most common etiologic agents of a number of illnesses including meningitis and nonspecific febrile illnesses. Recent reports have linked NPEV infection with chronic fatigue syndrome (Clements et al. J. Med. Virol. 45:156-161(1995).
In developed countries, polioviruses have been controlled with the introduction of vaccines in the late 1950""s. Vaccines typically contain either inactivated poliovirus, which is administered parenterally or live attenuated poliovirus, which is administered orally. The inactivated vaccines use tissue culture-derived poliovirus which has been inactivated, or killed with formaldehyde. Attenuated virus vaccines are prepared by passage of the virus in cell cultures until it loses its ability to cause the disease. Attenuated live virus replicates in the gut to induce a protective antibody response.
Virus used for these vaccines is typically cultured in African Green Monkey kidney cells. As noted above, a number of poorly characterized enteroviruses have been isolated from primates, including monkeys. Procedures are currently in place to identify monkey cells infected by other viruses (e.g., SV40) before use in culturing polioviruses.
Understanding how these molecular changes lead to disease is not well understood in the art. Increased understanding of the cellular mechanisms, particularly changes in nucleic acids, that occur early in the pathogenesis of these diseases is important to development of useful therapies and diagnostic tools. In addition, identification of viruses, including enteroviruses, in polio vaccine preparations is important to ensure safety of polio vaccines. Moreover, the possibility that new viruses resulting from recombination of poliovirus with other viruses from the monkey cells or the human gut is an obvious public health concern. The present invention addresses these and other concerns.
The present invention provides methods of screening for a disease state in a patient. The methods comprise providing a sample containing biological material (e.g., biopsies) or biological fluids from the patient (e.g., an acellular biological fluid such as serum or plasma) and contacting the sample with a nucleic acid which specifically hybridizes to a target nucleic acid sequence. The target nucleic acids are then detected. In some embodiments, the target nucleic acid includes sequences from a fragile site in the human genome, in particular, repetitive DNA. In some embodiments the target sequences are derived from Alu sequences in a fragile site. In other embodiments, the target nucleic acid may be a novel composite of microbial origin and in some cases human origin. The target nucleic acid is usually at least about 100 nucleotides in length, sometimes between about 500 and about 1500 nucleotides in length.
The methods are usually used to detect a chronic illness. Examples of chronic illnesses include cancers, such as multiple myeloma. Other diseases include autoimmune diseases, neurodegenerative diseases, heart diseases and the like.
In certain preferred embodiments, the target human nucleic acids are amplified (e.g., by PCR). An exemplary target sequence is provided in SEQ ID NO:23. This sequence can be used in diagnosis of multiple myeloma.
The present invention further provides improved methods for detecting viral nucleic acids in biological samples and polio vaccine preparations. In one embodiment, the invention provides methods for detecting recombinant viral nucleic acids, which comprise nucleic acid sequences from a polio virus and a non-poliovirus, usually a non-polioenterovirus. The methods comprise contacting a biological sample suspected of containing the recombinant viral nucleic acid with a first primer which specifically hybridizes to a conserved sequence in a picornaviral genome and a second primer which specifically hybridizes to a poliovirus nucleic acid sequence. The presence of an amplified product which is a recombinant viral nucleic acid is then detected.
A number of primers may be used in the present invention. For instance, one or both the primers may be one that specifically hybridizes to a 5xe2x80x2 nontranslated region of an picornaviral genome. Since the 5xe2x80x2 nontranslated region is conserved among picornaviruses, the primer will specifically hybridize to most picornaviruses, particularly enteroviruses. Primers PG01 and PG02 (as shown in SEQ ID NO:1 or SEQ ID NO:2 are conveniently used for this purpose. One or both of the primers may specifically hybridize to a P2-P3 region of a poliovirus genome. A preferred primer is one that specifically hybridizes to nucleotides 4922-4941 or nucleotides 5467-5487. Primers PG03 and PG04 (as shown in SEQ ID NO:3 or SEQ ID NO:4) are conveniently used for this purpose. One or both of the primers may also specifically hybridize to a P2 region of a poliovirus genome. A preferred primer is one that specifically hybridizes to nucleotides 4460-4478 or nucleotides 4634-4653. Primers PG07 and PG08 (as shown in SEQ ID NO:5 or SEQ ID NO:6) are conveniently used for this purpose. A preferred combination of primers is PG02 and PG03.
The methods may be carried out using a number of biological samples commonly used for clinical analysis of nucleic acids. A convenient sample is human serum, plasma, or white blood cells.
A number of methods may be used to detect the presence of the recombinant viral nucleic acid. In some embodiments, the detection is carried out using gel electrophoresis to identify an amplified fragment that is not present in a control sample known to contain only poliovirus nucleic acids. When the first primer selectively hybridizes to nucleotides 443-460 of a poliovirus genome (e.g., PG02) and the second primer selectively hybridizes to nucleotides 4922-4941 of a poliovirus genome (e.g., PG03) an amplified fragment of about 400 nucleotides in length can be used to detect the presence of a recombinant viral nucleic acid.
The invention also provides methods for detecting nonpoliovirus nucleic acids in a polio vaccine sample. The methods comprise contacting the vaccine sample with at least two primers which specifically hybridize to poliovirus nucleic acid sequences.
In these methods, one primer can be one that specifically hybridizes to a conserved sequence in an enteroviral genome, such as the 5xe2x80x2 nontranslated region. Exemplary primers include those that specifically hybridize to nucleotides 163-178 or nucleotides 443-450. Such primers include PG01 and PG02 (as shown in. SEQ ID NO:1 and SEQ ID NO:2).
A primer can also be one that specifically hybridizes to a sequence specific to a poliovirus genome, such as P2-P3 region of a poliovirus genome, for example, nucleotides 4922-4941 or nucleotides 5467-5487. Such primers include PG03 and PG04 (as shown in SEQ ID NO:3 and SEQ ID NO:4).
A primer can also be one that specifically hybridizes to a sequence specific to a poliovirus genome, such as the P2 region of a poliovirus genome, for example, nucleotides 4460-4478 or nucleotides 4634-4653. Such primers include PG07 and PG08 (as shown in SEQ ID NO:5 and SEQ ID NO:6).
In these methods, nonpoliovirus nucleic acids may be detected using gel electrophoresis to identify an amplified fragment that is not present in a control vaccine sample known to contain only poliovirus nucleic acids.
The invention further provides nucleic acid molecules from new, recombinant viruses identified here. The claimed molecules can be identified by their ability to hybridize to the exemplified sequences under stringent conditions, as defined below. The nucleic acids may be a complete viral genome, or fragments thereof. The nucleic acids may be isolated from a biological sample and may or may not be integrated in human chromosomal DNA.
Definitions
An xe2x80x9cacellular biological fluidxe2x80x9d is a biological fluid which substantially lacks cells. Typically, such fluids are fluids prepared by removal of cells from a biological fluid that normally contains cells (e.g., whole blood). Exemplary processed acellular biological fluids include processed blood (serum and plasma), urine, saliva, sweat, tears, phlegm, cerebrospinal, semen, feces and the like.
An xe2x80x9carchived nucleic acid sequencexe2x80x9d is a chimeric sequence in human genomic DNA containing subsequences from other organisms, particularly pathogens such as bacteria (e.g., members of the genera Chlamydia, Mycoplasma, Neisseria, Treponema, Staphylococcus, Streptococcus, and the like), parasites (e.g., Plasmodium falciparum, Pneumocystis carinii, Trichomonas, Cryptosporidium), viruses (e.g., herpes viruses, enteroviruses, polyoma viruses, poxviruses, such as Molluscum contagiosum viruses, retroviruses, such as HIV, and the like). Thus, when designing nucleic acids (e.g., as probes or PCR primers) for detecting archived nucleic acids of the invention, sequences based on the genome of these pathogens are conveniently used. Without wishing to be bound by theory, it is believed that archived nucleic acid sequences are usually inserted at fragile sites.
The term xe2x80x9cbiological samplexe2x80x9d, as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a xe2x80x9cclinical samplexe2x80x9d which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
A xe2x80x9cchronic illnessxe2x80x9d is a disease, symptom, or syndrome that last for months to years. Examples of chronic illnesses include cancers (e.g., multiple myeloma, leukemia, breast cancer, ovarian cancer, head and neck cancer, brain cancer, cervical cancer, testicular cancer, prostate cancer, Hodgkins Disease, and the like), precancerous conditions ( e.g., adenomatous polyposis coli (APC)), chronic fatigue syndrome, autoimmune diseases (e.g., arthritis, multiple sclerosis, lupus, scleroderma, and the like) diabetes, asthma, heart disease, neuromuscular diseases (e.g., fibromyalgia), neurodegenerative diseases (e.g., ALS, Alzheimer""s Disease, and Parkinson""s Disease), AIDS, Persian Gulf War Related Illnesses and chronic hepatitis.
A xe2x80x9cfragile sitexe2x80x9d is a locus within the human genome that is a frequent site of DNA strand breakage. Fragile sites are typically identified cytogenetically as gaps or discontinuities as a result of poor staining. Fragile sites are classified as common or rare and further divided according to the agents used to induce them. For a general description of fragile sites and their classification, see, Sutherland GATA 8:1961-166 (1991). Exemplified sequences disclosed here include sequences from viral genomes that have apparently been inserted into the human genome at a fragile site. Thus, fragile sites can contain xe2x80x9carchived nucleic acid sequencesxe2x80x9d which result from a wide range of pathogens, including bacteria, parasites, and viruses.
A xe2x80x9ctarget human nucleic acidxe2x80x9d of the invention is a nucleic acid molecule derived from human genomic DNA (e.g., chromosomal DNA, mitochondrial DNA, and other extrachromosomal DNA). As used herein human genomic DNA refers to germline DNA and may also include nucleic acids introduced into the individual as a result of infection of the individual by a pathogenic microorganism (e.g., exogenous viral DNA integrated into the genome after infection or through live virus infection). Thus, although target human nucleic acids of the invention are of human origin, they may nonetheless contain sequences shared by other pathogenic organisms, such as viruses. Such sequences are sometimes referred to here as human/viral chimeric sequences or xe2x80x9carchived sequencesxe2x80x9d. DNA xe2x80x9cderived fromxe2x80x9d human genome DNA includes DNA molecules consisting of subsequences of the genomic DNA as well as RNA molecules transcribed from human genomic DNA.
The RNA molecules detected in the methods of the invention may be free, single or double stranded, molecules or complexed with protein. Such RNA molecules need not be transcribed from a gene, but can be transcribed from any sequence in the chromosomal DNA. Exemplary RNAs include small nuclear RNA (snRNA), mRNA, tRNA, and rRNA.
The terms xe2x80x9chybridize(s) specificallyxe2x80x9d or xe2x80x9cspecifically hybridize(s)xe2x80x9d refer to complementary hybridization between an oligonucleotide (e.g., a primer or labeled probe) and a target sequence. The term specifically embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired priming for the PCR polymerases or detection of hybridization signal.
xe2x80x9cNucleic acidxe2x80x9d refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
The term xe2x80x9coligonucleotidexe2x80x9d refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, such as primers, probes, nucleic acid fragments to be detected, and nucleic acid controls. The exact size of an oligonucleotide depends on many factors and the ultimate function or use of the oligonucleotide.
The term xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligodeoxyribonucleotide sequence. The appropriate length of a primer depends on the intended use of the primer but typically ranges from about 15 to about 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to specifically hybridize with a template.
xe2x80x9cProbexe2x80x9d refers to an oligonucleotide which binds through complementary base pairing to a subsequence of a target nucleic acid. It will be understood by one of skill in the art that probes will typically substantially bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are typically directly labeled (e.g., with isotopes or fluorescent moieties) or indirectly labeled such as with digoxigenin or biotin. By assaying for the presence or absence of the probe, one can detect the presence or absence of the target.
The term xe2x80x9cregulatory sequencexe2x80x9d refer to cis-acting sequences (either 5xe2x80x2 or 3xe2x80x2) necessary for efficient transcription of structural sequences (e.g., open reading frames). These sequences include promoters, enhancers and other sequences important for efficient transcription and translation (e.g., polyadenylation sites, mRNA stability controlling sequences and the like).
A xe2x80x9csequence specific toxe2x80x9d a particular virus species or strain (e.g., poliovirus) is a sequence unique to the species or strain, that is, not shared by other previously characterized species or strains. A probe or primer containing a sequence complementary to a sequence specific to a virus will typically not hybridize to the corresponding portion of the genome of other viruses under stringent conditions (e.g., washing the solid support in 2xc3x97SSC, 0.1% SDS at about 60xc2x0 C., preferably 65xc2x0 C. and more preferably about 70xc2x0 C.).
The term xe2x80x9csubstantially identicalxe2x80x9d indicates that two or more nucleotide sequences share a majority of their sequence. Generally, this will be at least about 90% of their sequence and preferably about 95% of their sequence. Another indication that sequences are substantially identical is if they hybridize to the same nucleotide sequence under stringent conditions (see, e.g., Sambrook et al., Molecular Cloningxe2x80x94A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1985). Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.2 molar at pH 7 and the temperature is at least about 60xc2x0 C. For example, a nucleic acid of the invention or fragment thereof can be identified in standard filter hybridizations using the nucleic acids disclosed here under stringent conditions, which for purposes of this disclosure, include at least one wash (usually 2) in 0.2xc3x97SSC at a temperature of at least about 60xc2x0 C., usually about 65xc2x0 C., sometimes 70xc2x0 C. for 20 minutes, or equivalent conditions.
As used herein a xe2x80x9cviral nucleic acidxe2x80x9d is a nucleic acid molecule comprising nucleic acid sequences derived from viruses. Since as described below, the viral nucleic acids disclosed here are thought to be derived from recombination events, the viral nucleic acids of the invention may contain sequences derived from other microorganisms or from cellular sequences.
A nucleic acid comprising a xe2x80x9ccomplete viral genomexe2x80x9d is a nucleic acid molecule encoding all the polypeptide products required to construct a complete, infectious viral particle. For instance, in the case of enteroviruses, a complete viral genome would be a nucleic acid encoding all the protein products identified in FIG. 1. As used herein a complete, infectious viral particle can be encoded by a sequence that is a full length genome, as well as a substantially full length (e.g., 90%, preferably 95% complete) genome.